An S-Type Anion Channel SLAC1 Is Involved in Cryptogein-Induced Ion Fluxes and Modulates Hypersensitive Responses in Tobacco BY-2 Cells

Pharmacological evidence suggests that anion channel-mediated plasma membrane anion effluxes are crucial in early defense signaling to induce immune responses and hypersensitive cell death in plants. However, their molecular bases and regulation remain largely unknown. We overexpressed Arabidopsis SLAC1, an S-type anion channel involved in stomatal closure, in cultured tobacco BY-2 cells and analyzed the effect on cryptogein-induced defense responses including fluxes of Cl− and other ions, production of reactive oxygen species (ROS), gene expression and hypersensitive responses. The SLAC1-GFP fusion protein was localized at the plasma membrane in BY-2 cells. Overexpression of SLAC1 enhanced cryptogein-induced Cl− efflux and extracellular alkalinization as well as rapid/transient and slow/prolonged phases of NADPH oxidase-mediated ROS production, which was suppressed by an anion channel inhibitor, DIDS. The overexpressor also showed enhanced sensitivity to cryptogein to induce downstream immune responses, including the induction of defense marker genes and the hypersensitive cell death. These results suggest that SLAC1 expressed in BY-2 cells mediates cryptogein-induced plasma membrane Cl− efflux to positively modulate the elicitor-triggered activation of other ion fluxes, ROS as well as a wide range of defense signaling pathways. These findings shed light on the possible involvement of the SLAC/SLAH family anion channels in cryptogein signaling to trigger the plasma membrane ion channel cascade in the plant defense signal transduction network.药理学研究证据表明，阴离子通道介导的质膜阴离子外流在植物早期防御信号通路中发挥关键作用，可诱导免疫应答与超敏细胞死亡。然而，其分子机制与调控方式目前仍未被完全阐明。本研究在烟草悬浮培养BY-2细胞中过表达参与气孔关闭的拟南芥S型阴离子通道SLAC1，并分析其对隐地蛋白诱导的防御应答的影响，包括Cl⁻与其他离子的流动、活性氧（ROS）生成、基因表达与超敏反应。SLAC1-GFP融合蛋白在BY-2细胞中定位于质膜。SLAC1过表达可增强隐地蛋白诱导的Cl⁻外流与胞外碱化，同时促进NADPH氧化酶介导的ROS生成的快速瞬时相与缓慢持续相，该效应可被阴离子通道抑制剂DIDS所抑制。过表达细胞系还对隐地蛋白表现出更高的敏感性，可诱导下游免疫应答，包括防御标记基因的表达与超敏细胞死亡。上述结果表明，在BY-2细胞中表达的SLAC1可介导隐地蛋白诱导的质膜Cl⁻外流，从而正向调控激发子触发的其他离子流动、ROS生成以及多种防御信号通路的激活。本研究结果揭示，SLAC/SLAH家族阴离子通道可能参与隐地蛋白信号通路，从而在植物防御信号转导网络中触发质膜离子通道级联反应。